Scores of neutrophilic infiltration of alveolar septa in BRSV-infected calves.

<p>HE stained sections. a) score 0 (vaccinated calf a, experiment I), b) score 1 (vaccinated calf b, experiment I), c) score 2 (non-vaccinated, BRSV-infected calf l, experiment I) and d) score 3 (non-vaccinated, BRSV infected calf D5, experiment II). Lesions are examples of neutrophil scores and not representative for the overall inflammation regarding exudate and consolidation.</p

Detection of citrullinated histone 3 and myeloperoxidase in serially diluted BAL.

<p>Detection of citrullinated histone 3 (a) and myeloperoxidase (b) by dotblot in bronchoalveolar lavage (BAL) from BRSV-infected calves with clinical signs of disease and high levels of virus shedding (k-o and D1-D5) compared to that in BRSV-ISCOM-vaccinated, BRSV-infected calves with no or little clinical signs of disease and no or low levels of virus shedding (a-e, Experiment I), or non-vaccinated, non-infected calves (E1-E5, Experiment II). The proteins were selected based on being present in neutrophil extracellular traps, incriminated to be important in the pathogenesis of RSV [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186594#pone.0186594.ref030" target="_blank">30</a>].</p

Animals and experimental design.

<p>Animals and experimental design.</p

Correlations (Spearman’s rho) between relative expression level of selected proteins in bronchoalveolar lavage from calves six days post BRSV infection, BRSV-induced disease and presence of neutrophils in alveolar septa.

<p>Correlations (Spearman’s rho) between relative expression level of selected proteins in bronchoalveolar lavage from calves six days post BRSV infection, BRSV-induced disease and presence of neutrophils in alveolar septa.</p

Semi-quantification of selected proteins involved in processes that were affected by BRSV-infection.

<p>Proteins were detected in bronchoalveolar lavage from non-vaccinated, BRSV-infected calves with clinical signs of disease and high levels of virus shedding (black bars) and/or in BRSV-ISCOM-vaccinated, BRSV-infected calves with no or little clinical signs of disease and no or low levels of virus shedding (a, experiment I, grey bars), and/or non-vaccinated, non-infected calves (b, experiment II, grey bars). Proteins were identified by LC-MS/MS and semi-quantified by label-free analysis. Statistically significant differences are indicated by asterisks; p≤0.05(*); p≤0.01 (**); p≤0.001 (***). Proteins were selected based on being related to neutrophil activation and chemotaxis or detoxification of reactive oxygen species: biological processes identified by protein pathway analysis.</p

Proteins associated with BRSV-induced disease.

<p>Proteins associated with BRSV-induced disease.</p

Bronchoalveolar proteomes of calves.

<p>Proteins were identified by LC-MS/MS in bronchoalveolar lavage of calves and were classified as detected at group level if identified in at least 4 out of 5 calves per group. The groups consisted of non-vaccinated, BRSV-infected calves with clinical signs of disease and high levels of virus shedding (a and b, right circles), or ISCOM-vaccinated, BRSV-infected calves with no or little clinical signs of disease and no or low levels of virus shedding (a, experiment I, left circle), or non-vaccinated, non-infected calves (b, experiment II, left circle). Protein names are provided as supplemental data (*) or in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0186594#pone.0186594.t001" target="_blank">Table 1</a> (**).</p

RSV proteins and epitopes used in subunit vaccine formulation.

a<p>Protein product included in subunit vaccine formulations, as abbreviated in the current paper.</p>b<p>HRSV Long strain (GenBank accession no. AY911262) was used to construct recombinant protein.</p>c<p>Full length HRSV M2-1 protein (Long strain).</p>d<p>Full length HRSV P protein (Long strain).</p>e<p>BRSV strain 9402022 (Larsen et al. 1998) was used to construct recombinant protein.</p>f<p>Selected residues were recombinantly attached to the N or C terminus of HRSV N protein (Long strain) and co-expresses with a fragment of HRSV P protein (Long strain, AA residues 161–241) to form N-nanorings with attached epitopes (eN) on each of 10 or 11 protomers.</p>g<p>Antigenic site II (AA residues 422–438) on F, as described by Chargelegue et al. (1998).</p

Vaccination reduces the extent of lung lesions following BRSV challenge.

<p>Four groups of 5 calves were vaccinated as described in Fig. 1 and challenged with BRSV, 5 weeks after vaccination. Two weeks before challenge, one calf (c5) was euthanized due to traumatic injury. Lungs were removed after exsanguination, lesions were recorded on a lung chart after visual examination and palpation, and the proportion of lung showing pneumonic consolidation was calculated. Formalin-fixed tissue samples from each lobe in the right lung were analyzed for the severity of histopathological changes and scored as either normal (0), mild (1), moderate (2) or severe (3). (A) shows the extent of macroscopic lesions on the y-axes, and the microscopic severity of inflammation (mean score of four sections per calf) on the x-axes. Statistically significant difference is indicated by asterisks (p≤0.05). (B) shows the percent of pneumonic consolidation in each animal (also depicted as filled areas in lung-charts), and emphysema (outlined areas in calves d3 and d4). Panels C (I–IV) show representative histological images from each of the four groups of calves. Bar indicate 100 µm. Panels C (I) (ΔSHrBRSV), C (II) (SUMont), C (III) (SUAbis) and C (IV) (Control) show lung parenchyma with minimal, mild, moderate and severe pathological changes, respectively.</p

Experiment timeline, vaccination and sampling.

<p>Twenty calves with moderate titers of BRSV-specific serum antibodies (MDA) were allocated into 4 groups and vaccinated as indicated in the figure; all were vaccinated on post-vaccination day (PVD) 0 (Vacc. I, white arrow) with either (a) 5×10⁶ pfu of ΔSHrBRSV intranasally (i.n.); (b) BRSV and HRSV recombinant protein subunits (SU) adjuvanted by Montanide (SUMont) intramuscularly (i.m.), (c) SU adjuvanted by AbISCO-300 (SUAbis) subcutaneously (s.c.), or (d) adjuvant alone s.c. (Controls). On PVD 20, all animals except those immunized with ΔSHrBRSV, were boosted with the same formulation and route as for Vacc. I (Vacc. II, gray arrow). Three BRSV-seronegative calves were housed in contact with ΔSHrBRSV-infected animals to determine transmission of the vaccine virus (Sentinel calves), and monitored until euthanized (†) on PVD 22. On PVD 20, one calf in group c was euthanized due to traumatic injury. On post-infection day (PID) 0, all calves were challenged i.n. with 10⁴ pfu virulent BRSV (black arrow), and clinically scored daily until PID 7. Throughout the experiment, samples were collected, as indicated in the figure, to analyze antibodies in serum and nasal secretions, <i>ex-vivo</i> response of peripheral blood mononuclear cells (PBMC) to restimulation with BRSV, and virus shedding in nasal secretions (Nasal swab). At post-mortem examination (PM), lung lesions were recorded and tissue samples collected, as well as bronchoalveolar lavage (BAL) samples for antibody, BRSV RT-PCR and virus isolation.</p